Project Summary/Abstract In normal physiology, the repertoire of human T cells includes a 10% subpopulation that expresses two, rather than a single, T cell receptor (TCR). Despite the obvious implications of dual-specificity at the clonal level, the function of this dual TCR subpopulation remains largely unknown. We recently proposed the novel paradigm and provided evidence demonstrating that naturally-arising dual TCR expression is important in thymopoiesis and that dual TCR cells have exceptional ability to recognize ligands driving alloreactivity and autoreactivity. This reactivity is relevant to disease, as we demonstrated in mouse models and human patients that dual TCR T cells are important drivers in graft versus host disease, a severe and life-threatening T cell-mediated complication of hematopoietic stem cell transplantation. Data from other models also link dual TCR T cells to autoimmune disease such as diabetes. Despite evidence of the importance of dual TCR cells, they have been understudied due to the inability to definitively identify, isolate, and functionally study T cells expressing dual TCRs. To address this, we have developed novel approaches including single-cell TCR sequencing of mouse and human cells, and transgenic mice with fluorescent reporters for TCR?. We intend that these tools will enable us to address our hypotheses and generate novel insights into fundamental TCR biology to identify potential avenues to improve clinical transplantation.